Genetically modified mice in reproduction.

Question 1

List 8 reasons why we use in vivo models.

Answer 1

• Whole body homeostasis
• The phenomenon to be studied does not occur in vitro
• The cell type to be studied cannot be cultured (e.g. highly differentiated)
• Cell type-to-cell type or tissue-to-tissue interactions
• Different stages of developmental
• Endocrine systems typically involve multiple organ interactions
• Drug discovery
• Best proof-of-concept tool available.

Question 2

List the advantages of using the mouse as an animal model.

Answer 2

• Controlled environment
• Experimental interventions possible
• Fast reproductive cycle
• Genome sequenced
  -Many genetic strains and mutants described
  -A well characterized animal model relatively
  close to humans physiology
• Many kinds of genetic manipulations possible (add, delete, mutate)

Question 3

List the disadvantages to using the mouse as an animal model.

Answer 3

• Long lifespan
• Expensive
• Size
• Ethical concerns require good justification
• Large genome (difficult to screen certain manipulations)
• Concerns about the relevance to human diseases
• Multigenic pathologies difficult to study.

Question 4

What are the applications of genetically modified mice?

Answer 4

• Study of transcriptional regulatory elements: gene regulation studies with reporter genes
• Examining consequences of overexpression of a gene: universal or tissue/cell-specific over-expression
• Analysing consequences of expressing a dominant negative mutation: overexpressing an abnormal gene, universal or tissue/cell specific knockout approaches.

Question 5

What are the techniques used for genetically modified mice?

Answer 5

1) Injection of transgene into pronucleus- germ line modification
2) Injection of ES cells engineered to contain gene/mutation into blastocyst.

Transferred to pseudo-pregnant female.

Screened for genotype to determine if carry gene/mutation introduced.

Question 6

Outline the principles of genetic modification of the germ-line.

Answer 6

• Germ cells are modified with transgene of interest- pronuclear injection, post fertilisation, before fusion.
• Transmission of the transgene through chromosomal integration and embryonic divisions to the next generation as any other chromosomal gene.
• All cells of resulting transgenic animal carry the same modification
• Usually hemizygous. Each offspring may have different copy number therefore treated as different line.

Question 7

Give examples of genetic modification of the germ-line.

Answer 7

1) Global overexpression/knockout mice for research tools
2) TG pigs for xenotransplantation donors
3) “Humanisation” of animals expressing human genes- first TG mouse expressing hGH.

Question 8

Outline the principles of embryonic stem cell modification.

Answer 8

• The transgene is introduced into embryonic stem (ES) cells.
• ES cells that have undergone homologous recombination are identified and injected into a 4 day old mouse embryo - a blastocyst.
• Background of ES cells and blastocyst are usually different-white and black mice- chimeric animals.
• Modify an existing genomic locus: knock-out, knock-in, partial deletion etc.

Question 9

Describe other genetics modification ideas.

Answer 9

• Cre-loxP: tissue specific knock in/out
• Inducible: drug-inducible promoters (e.g. tetracyclin and tamoxifen) allowing temporal expression/deletion of a transgene (e.g. Cre - to induce KO).
• Reporter gene: firefly luciferase, b-gal, eGFP, mcherry, ROSA, IRES. Spatial/temporal information.
• BACS: integrate entire gene (including reg elements, promoter).
• CRISPR/Cas9- guide RNA and modifying Cas9 endonuclease- gene editing.

Question 10

What happens in a human male with a LH/LHR inactivation?

Answer 10

• pseudohermaphroditism (LHR)
• normal sexual differentiation (LHβ)
• Leydig cell hypoplasia
• lack of pubertal maturation

Question 11

What happens in a human female with a LH/LHR inactivation?

Answer 11

• normal sexual differentiation
• delayed pubertal maturation
• oligomenorrhea/amenorrhea
• anovulatory infertility

Question 12

What happens in a mouse male with a LH/LHR inactivation?

Answer 12

• normal sexual differentiation
• Leydig cell hypoplasia
• lack of pubertal maturation
• infertility

Question 13

What happens in a mouse female with a LH/LHR inactivation?

Answer 13

• normal sexual differentiation
• delayed vaginal opening
• no estrous cycle
• anovulatory infertility

Question 14

What is the backup mechanism in the human for male masculinisation if there is a LH mutation?

Answer 14

hCG can act of LHR encouraging testosterone production and therefore male differentiation.

Question 15

What is the backup mechanism in the mouse for male masculinisation if there is an LH and LHR mutation?

Answer 15

Paracine factors act on the LHR encouraging testosterone production and therefore male differentiation.

Question 16

What happens in the human male if there is an LHR mutation?

Answer 16

No testosterone production and no male differentiation occurs.

Question 17

What are the consequences of an activating mutation of FSHR?

Answer 17

• Men: poor spermatogenesis

* Women: hypergonadotrophic hypogonadism

Question 18

What are the consequences of an inactivating mutations of FSHR?

Answer 18

• Men: spermatogenesis without gonadotrophins in a hypophysectomized man (n = 1)
• Women: no real activating mutations detected, only mutations altering ligand specificity (->hCG) -> pregnancy-associated ovarian hyper stimulation
• Accelerated ovarian ageing-> accumulation of lipofuscin pigment and collagen.

Question 19

What are the consequences of CAM-FSHR in female mice?

Answer 19

• Hemorrhagic cysts
• Depletion of follicles/POF (Prem ovarian insufficiency)
• Luteinized unruptured follicles
• Teratomas.
(Higher Oestrogen levels)